Bull. Soc. Pathol. Exot. DOI 10.1007/s13149-010-0044-1

EPIDEMIOLOGY / EPIDÉMIOLOGIE

Dynamics of Leishmania infection rates in Rhombomys opimus (Rodentia: Gerbillinae) population of an endemic focus of zoonotic cutaneous leishmaniasis in Dynamique des taux d’infection à Leishmania chez les populations de Rhombomys opimus (Rodentia : Gerbillinae) dans un foyer endémique de leishmaniose cutanée zoonotique en Iran

A.A. Akhavan · M.R. Yaghoobi-Ershadi · A. Khamesipour · H. Mirhendi · M.H. Alimohammadian · Y. Rassi · M.H. Arandian · R. Jafari · H. Abdoli · N. Shareghi · M. Ghanei · N. Jalali-zand

Received: 2 November 2009, Accepted: 23 December 2009 © Société de pathologie exotique et Springer-Verlag France 2010

Abstract Zoonotic cutaneous leishmaniasis (ZCL) due to reaction (PCR) assay was used for the detection and identi- is a great public health problem in the fication of Leishmania parasites, and the results were con- Old World. Leishmania major is widely distributed in popu- firmed by PCR–restriction fragment length polymorphism lations of in arid and savannah regions. In this study, (RFLP). The results showed that Leishmania infection rate seasonal variation of natural infection with Leishmania para- was 55.8% (29 out of 52 gerbils) using nested PCR. The sites in Rhombomys opimus (Rodentia: Gerbillinae) popula- highest and lowest Leishmania infection rates were observed tion of an endemic focus of ZCL in Iran was monitored. The in fall and summer, respectively. Gerbils that were found to study was conducted from October 2007 to October 2008 in be infected only with L. major were 5.8%, and that with the central part of the country. Nested polymerase chain Leishmania turanica were 23.1%. A mixed natural infection was seen in the rodents with L. major and L. turanica (21.2%), with L. major and L. gerbilli (1.9%), and with all A.A. Akhavan · M.R. Yaghoobi-Ershadi (*) · Y. Rassi the three species (3.9%). Leishmania major infection alone Department of Medical Entomology and Vector Control, was seen in fall and winter whereas mixed infection of School of Public Health, Tehran University of Medical Sciences, L. major and L. turanica was observed in all seasons except Tehran, Iran in summer. Leishmania turanica infection was observed e-mail : [email protected] throughout the year. It is concluded that L. major, L. gerbilli, A. Khamesipour and L. turanica circulate in the population of R. opimus in Center for Research in Skin diseases and Leprosy, central part of Iran. Leishmania major infection is usually Tehran University of Medical Sciences, accompanied by L. turanica in naturally infected gerbils Tehran, Iran with the highest rate in fall. It is recommended that the role H. Mirhendi of L. turanica in the epidemiology and transmission of ZCL Department of Medical Parasitology and Mycology, should be reconsidered. School of Public Health, Tehran University of Medical Sciences, Tehran, Iran Keywords Rhombomys opimus · Leishmania major · L. gerbilli · L. turanica · Seasonal variation · Zoonotic M.H. Alimohammadian cutaneous leishmaniasis · Nested PCR · Badrood · Iran · Department of Immunology, Pasteur Institute of Iran, Middle South Asia Tehran, Iran

M.H. Arandian · R. Jafari · H. Abdoli · N. Shareghi · M. Ghanei · Résumé La leishmaniose cutanée zoonotique (LCZ) due à N. Jalali-zand Leishmania major constitue un problème de santé publique Esfahan Training and Health Research Center, dans l’ancien monde. L. major est une espèce largement dis- National Institute of Health Research, Tehran University of Medical Sciences, tribuée dans les populations de rongeurs vivant dans les Esfahan, Iran régions arides et de savane. Dans la présente étude, la 2 Bull. Soc. Pathol. Exot. variation saisonnière de l’infection naturelle par les parasites Iran; obesus () and cras- du genre Leishmania chez la Grande Gerbille Rhomb- sus in northern Africa and Middle East; and Tatera spp. in omys opimus (Rodentia: Gerbillinae) au sein d’une popula- subsaharan Africa and Iran [6]. R. opimus (Cricetidae: Gerbil- tion d’un foyer endémique de LCZ en Iran a été contrôlée. linae) is the principal L. major reservoir host in the vast terri- L’étude a été menée d’octobre 2007 à octobre 2008 dans la tory of the Turan lowland (west and south and partie centrale de l’Iran. Une PCR nichée a été utilisée pour with adjacent parts of and Iran), la détection et l’identification du parasite Leishmania, et les and, apparently, in some provinces of . In résultats ont été confirmés par PCR-RFLP. Les résultats ont the Turan lowland, naturally infected R. opimus were reported montré que le taux d’infection par Leishmania était de from more than 200 places. The number of naturally infected 55,8 % (29 sur 52 animaux) en utilisant la PCR nichée. great gerbils showed to be greater than any other Les taux d’infection par Leishmania les plus forts et les (other rodents, insectivores, and carnivores) [3]. plus faibles ont été observés, respectivement, en automne All the proven vectors of ZCL belong to the subgenus Phle- et en été ; 5,8 % des gerbilles étaient infectées par L. major botomus,i.e.Phlebotomus papatasi, the principal vector, and isolément et 23,1% par L. turanica isolément. Une infection related species: Phlebotomus salehi and Phlebotomus dubosqi. mixte naturelle a été observée chez les rongeurs associant Well-described stable ZCL systems are associated with L. major et L. turanica (21,2 %), ou associant L. major et L. major, Psammomys obesus,andPh. papatasi in North L. gerbilli (1,9 %), ou associant les trois espèces (3,9 %). Africa and Middle East and with R. opimus and Ph. papatasi L’infection due à L. major isolément a été observée en auto- in central Asia, Afghanistan, and Iran [6,19]. The distribution mne et en hiver. Une infection mixte associant L. major et and the role of rodents as reservoir hosts of ZCL are geograph- L. turanica a été observée en toutes saisons, sauf en été. ically specific in Iran. Rhombomys opimus is the main reser- L’infection à L. turanica a été observée tout au long de l’an- voir of ZCL in Central and North East (N.E.) Iran followed by née. En conclusion, les trois espèces L. major, L. gerbilli et M. libycus (Cricetidae: Gerbillinae), which is the primary res- L. turanica circulent dans la population de R. opimus dans la ervoir of ZCL in some areas of the central and southern Iran. In partie centrale de l’Iran. L. major est habituellement accom- the south and south west of the country, including the Iran– pagnée par L. turanica chez les gerbilles naturellement Iraq border, the reservoir is Tatera indica, the Indian infectées avec un plus fort taux en automne. Le rôle de Jird (Cricetidae: Gerbillinae). In Baluchistan of Iran (border L. turanica dans l’épidémiologie et la transmission de la of ), M. hurrianae (Cricetidae: Gerbillinae) acts as a LCZ devrait être reconsidéré. reservoir host [7,8,12,13,18,22]. One of the major problems for the control and understanding of this neglected disease is Mots clés Rhombomys opimus · Leishmania major · the lack of information about the dynamics of Leishmania L. gerbilli · L. turanica · Variation saisonnière · parasites in rodent reservoir populations. In this study, sea- Leishmaniose cutanée zoonotique · PCR niche · Badrood · sonal variation of Leishmania species infection in Rhombomys Iran · Moyen-Orient opimus (Rodentia: Gerbillinae) population of an endemic focus in Central Iran was monitored.

Introduction Materials and methods

Zoonotic cutaneous leishmaniasis (ZCL), a neglected tropical Study area disease, is a public health problem with a clear and disturbing increase in the number of cases in some areas of the world The investigation was conducted over a period of 12 months [2,6]. Leishmania major is widely distributed in various from October 2007 to October 2008 in Badrood rural district populations of rodents in arid and savannah regions [6]. The (33° 42′ N, 52° 2′ E), 25 km from the city of Natanz, Natanz disease is endemic in many rural districts of Iran, in county, Esfahan Province, central Iran where ZCL is 17 provinces out of 30. Rodents belonging to subfamily Ger- endemic. Badrood district is located at an altitude of billinae are the main reservoir hosts of ZCL in Iran and other 1056 m, in the foothills of Karkas Mountains (altitude countries where ZCL is endemic [3,15,18]. Gerbils are the 3898 m) with a climate, hot summer and cold winter. most abundant mammals reported from natural ecosystems of Old World [3]. Many rodent species act as reservoir Rodent collection hosts of ZCL: Rhombomys opimus () in central Asia, northern Afghanistan, and Iran; Meriones libycus Active colonies of gerbils in the district were identified and () in the Arabian Peninsula, central Asia, and the rodents were caught using approximately 40 Sherman Iran; Meriones hurrianae () in and traps baited with cucumber, in different seasons. In spring Bull. Soc. Pathol. Exot. 3 to fall, the traps were placed at the gerbil holes in the after- were visualized by agarose gel electrophoresis and ethidium noon and were collected in the morning on the following bromide staining. The identity of all species identified by day. In winter, Sherman traps were placed after sunrise and nested PCR was confirmed based on species-specific pattern were collected at noon on the same day. The trapped gerbils of PCR-RFLP using the restriction digestion with MnlI. were transferred to the house facility at the Esfahan Digestion was performed by adding 5 U (0.5 μl of the Training and Health Research Center, National Institute of enzyme) and 1.5 μl of the relevant buffer to a 13 μl aliquot Health, Esfahan, Iran, and maintained until use for parasito- of the nested PCR product in a final volume of 15 μl. logical and molecular testing. The mixture was incubated at 37° C for 3 h and the products The rodents were identified using morphological charac- were separated using 2.5% agarose gel electrophoresis and ters [5] and only great gerbils, R. opimus, were used for the visualized by ethidium bromide staining. study.

Direct examination test Statistical analysis

In the laboratory, the rodents were anesthetized using intra- The chi-square and Fisher’s exact tests using SPSS 11.5 soft- muscular ketamine hydrochloride (60 mg/kg) and xylazine ware were used and P value of less than 0.05 was considered (5 mg/kg). Regardless of obvious lesions, impression smears significant. were prepared from the ear lobes of the [4], stained by Giemsa, and directly examined carefully under a light Results microscope at high magnification (1000×) to search for Leishmania parasites. After preparing direct smears, ear A total of 52 R. opimus (22 male and 30 female) were cap- lobe samples were removed while the rodents were anesthe- tured and examined by two diagnostic techniques, direct tized. The ear lobes were transferred to cold phosphate- examination and nested PCR. Twelve out of fifty-two speci- buffered saline (pH = 7.4) and thoroughly disrupted by mens (23.1%) were positive by microscopic examination grinding with a pestle and kept at -20° C until use. The ani- and 29 (55.8%) by the nested PCR. In 17 samples in which mals were nursed to complete recovery. Animal experiments the amastigote was not seen by direct examination, the were approved by the Ethics Committee of Tehran Univer- nested PCR showed a positive result, and every positive sity of Medical Sciences, Tehran, Iran. smear was positive by nested PCR. Out of 29 positive sam- ples by nested PCR, 3 (10.3%) were L. major, 12 (41.4%) Nested PCR assay were L. turanica, 11 (37.9%) were mixed infection of L. major and L. turanica, 1 (3.5%) was L. major and Genomic DNA was extracted and purified using a conven- L. gerbilli, and 2 (6.9%) were mixed infection of all three tional phenol–chloroform protocol. Briefly, 200 μl of lysis species. The Leishmania infection rates of male and female buffer (100 mM Tris–HCl, pH = 8; 10 mM EDTA, pH = 8; gerbils were 63.6% and 50%, respectively, which was not 1% SDS; 100 mM NaCl; 2% Triton X-100) with proteinase statistically different. The highest (92.9%) and lowest K (100 mg/ml) were added to 100 μl homogenized suspen- (20%) infection rates of Leishmania were observed in fall sion of disrupted tissues and cells. The sample was incubated and summer, respectively (Fig. 1). Statistically significant at 56 °C for 1 h and subjected to phenol–chloroform extrac- difference was observed in infection rates of Leishmania tion (phenol–chloroform followed by chloroform). The among different seasons (P = 0.001). Around 90% of the DNA was precipitated with an equal volume of isopropanol infected gerbils showed no cutaneous leishmaniasis lesion and 1/10 volume of 3 M sodium acetate (pH = 5.2). The on their ear lobes. pellet was washed with 70% ethanol, air dried at room tem- Three out of fifty-two (5.8%) of the gerbils were found to perature, and resuspended in 20 μl of sterile distilled water. be infected only with L. major and twelve (23.1%) with Fragment length polymorphism of the second internal L. turanica.NoL. gerbilli infection alone was seen in the transcribed spacer(ITS2) in the ribosomal RNA gene gerbils. A mixed natural infection was seen in the rodents (rDNA) based on a nested PCR system was used for detec- with L. major and L. turanica (21.2%), with L. major and tion and species identification of Leishmania in the speci- L. gerbilli (1.9%), and with all three species (3.9%). Mixed mens. The sequence of the primers were as follows: Leish infection of L. gerbilli and L. turanica was not seen in this out F (5′-AAA CTC CTC TCT GGT GCT TGC-3′) and study. Pure L. major infection was only observed in fall and Leish out R (5′-AAA CAA AGG TTG TCG GGG G-3′)as winter whereas mixed infection of L. major and L. turanica the outer primers and Leish in F (5′- AAT TCA ACT TCG was shown in all seasons except summer. Infection with CGT TGG CC-3′) and Leish in R (5′-CCT CTC TTT TTT L. turanica was seen throughout the year. The highest infec- CTC TGT GC-3′) as the inner primers. The PCR products tion rate of Leishmania was seen with L. turanica (50%) and 4 Bull. Soc. Pathol. Exot. Taux Positive (%) L. gerbilli , Oct. 2008 / – L. turanica and No. of positive samples/no. of examined samples Positive (%) Fig. 1 Seasonal variation of infection rates of Leishmania of Great L. turanica L. major

Gerbil population, Badrood rural district, Esfahan Province, Iran, and October 2007–October 2008 / Variation saisonnière des taux d’in- fection par Leishmania chez les populations de Grande Gerbille, dans les districts ruraux de Badroo, province d’Esfahan, Iran, oct. No. of positive samples/No. of examined samples 2007 – oct. 2008 Positive (%) L. gerbilli L. major mixed infection of L. major and L. turanica (35.7%), which was observed in fall (Table 1). and

Discussion No. of positive samples/No. of examined samples

The results of the current study showed that L. major, L. gerbilli, and L. turanica circulate in R. opimus popula- selon les saisons, dans les districts ruraux de Badrood tions in the study region. To our knowledge, seasonal varia- Positive (%) tion of mixed natural infections of R. opimus with L. major, L. turanica, and L. gerbilli have not been reported earlier. Therefore, this is the first time that the dynamics of such No. of positive samples/no. of examined samples mixed infections have been investigated in wild R. opimus, population in different seasons, Badrood rural district, Esfahan Province, Iran, Oct. 2007 the Great Gerbil, in Iran or elsewhere. In spring, at the begin- ning of active season of sand flies [17], the highest Leish- Rhombomys opimus mania infection rate in R. opimus populations was due to L. turanica.TheinfectionrateofL. major was less at this Positive (%) time but higher in fall (Table 1). In Turkmenia and Uzbekistan,

epizootics among R. opimus populations always developed Rhombomys opimus with L. turanica at the beginning of transmission season.

TheinfectionrateofL. major was extremely less in June No. of positive samples/no. of examined samples chez les populations de and increased in late August and September [16]. In the district of Borkhar, an hyperendemic focus of the disease in Esfahan Province, in approximately 135 km from Badrood district, the Positive (%) highest infection rates of Leishmania in great gerbils were Leishmania reported from August to December [22]. For vast territories of central Asia, mixed infections of species infection rates of wild rodents with L. major (pathogenic to humans) and L. turanica (nonpathogenic to humans) are typical. Each L. major L. gerbilli L. turanica L. major No. of positive samples/no. of examined samples

parasite has its own range of pathogenicity and virulence Leishmania [15]. This animal proved to be susceptible to L. major, L. turanica, and L. gerbilli. Infection with L. major alone rarely infection par les espèces de

occurred in R. opimus. Leishmania turanica promotes the ’ d Season Species Tableau 1 persistence of L. major infection in the Great Gerbil [16]. SpringSummerFall 0/13 0/5WinterTotal 1/14 2/20 0 0 3/52 7.1 10 0/13 0/5 5.8 0/14 0/20 0 0/52 0 0 0 2/13 0 1/5 7/14 2/20 15.4 12/52 20 0/13 50 10 23.1 0/5 0/14 1/20 0 1/52 0 0 1/13 5 1.9 0/5 5/14 11/52 5/20 7.7 0/13 21.2 35.7 0 25 2/52 0/14 0 2/20 0/5 3.9 0 10 0 Bull. Soc. Pathol. Exot. 5

In an experimental L. major infection, the duration of the tance to carry out the project. This work was funded by disease was 7 months; in L. turanica infection, it was 15 Deputy of Research of Tehran University of Medical months; and in infection by L. gerbilli, it was 18 months. Sciences, through project no 3025. However, in coinfections of L. major and L. turanica, the disease duration was extended up to 39 months. Ulceration and visceralization never happened in great gerbils [14]. References Great gerbils remained as ZCL reservoir hosts for life and seemed to be potential sources of Leishmania transmission 1. Akhavan AA, Yaghoobi-Ershadi MR, Hasibi F, et al (2007) Emer- until death [15]. Leishmania turanica proved to be the do- gence of cutaneous leishmaniasis due to Leishmania major in a – minant species in R. opimus population located in hypoen- new focus of Southern Iran. Iranian J Arthropod-Borne Dis 1: 1 8 2. 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